Project Summary The goal of my research program is to understand the mechanisms underlying how microbial communities assemble in wound tissue and how these communities influence tissue repair pathways in the skin. During normal wound healing, the process that leads to tissue regeneration results from a series of tightly regulated sequential events. In the case of chronic, non-healing wounds, this process is disrupted, leading to a prolonged inflammatory response and stalled healing. Key recent advances in the understanding of impaired wound healing is the knowledge that complex microbial communities (a microbiome), exist within the wound tissue. Microbiomes in the wound tissue do not always cause clinical infection, but it is thought that they might sustain inflammation, further impairing healing pathways and skin repair. The majority of all published wound microbiome studies profile the taxonomic composition, but it is unclear how these diverse microbial communities actually interact with and influence host repair pathways. Thus, the extent of microbial influences on tissue regeneration remains to be resolved. The skin itself is a diverse ecosystem harboring beneficial microbial symbionts that function to regulate host immune responses and protect against pathogen colonization. We and others have shown that chronic wound microbiomes are comprised largely of skin commensals co-existing with skin pathogens and environmental organisms. This finding suggests that the benefits normally conferred by the skin microbiota may be lost in a wound environment, and it also highlights our incomplete understanding of how microbial interactions shape host health. The proposed research program will define the processes by which structured microbial communities form in wound tissue and will identify the molecular mechanisms governing inter-species interactions within these communities. We will accomplish this using a live ex vivo human skin wound model that we have developed to directly monitor growth dynamics of multi-species consortium and immune responses of resident skin cells. We can then determine how wound healing pathways shift in response to the makeup of the community and phenotypic traits, such as formation of microbial biofilm, in a porcine model of wound healing. We will also build upon our recent discovery that members of the healthy skin microbiota are exceptionally good at inhibiting the growth of diverse fungi. When placed in an ecological context, this finding is not surprising, as the skin has very low fungal diversity. However, we have also shown that fungal colonization and interactions with bacteria are significantly associated with necrosis of wound tissue and delayed healing. We will characterize the metabolites produced by the healthy skin microbiota mediating competitive interactions with fungi and determine their role in modulating microbial community dynamics both on healthy skin and within wound microbiomes containing fungi and bacteria in close interaction. Our studies will advance our understanding of impaired wound healing and will uncover fundamental processes governing dynamics of microbial community assembly.